Chromosomes are organized and separated during cell division by a microtubule-based molecular machine, the mitotic spindle. Our overall goal is to reconstitute spindle functions using pure components and apply new tools for manipulating and tracking single molecules to uncover how this machine operates. Here we request support for an ultrasensitive fluorescence microscope capable of simultaneously recording interactions between individual protein molecules, multiprotein complexes, and dynamic microtubule filaments. The instrument is based on an Olympus IX51 inverted microscope equipped with total internal reflection fluorescence (TIRF) illumination and with three Andor emCCD cameras, enabling fast, multicolor molecular tracking. It will be used by four NIH-funded investigators, each with complementary expertise, working in close collaboration with one another. The Davis lab will study super-complexes assembled from pure, recombinant forms of the six protein subcomplexes that make up kinetochores to determine how their interactions allow kinetochores to link chromosomes to spindle microtubules. The Asbury and Biggins labs will take a complementary approach, studying whole kinetochores isolated from wild-type yeast and from mutants with targeted defects to assess quantitatively the contribution each kinetochore subcomplex makes to chromosome-microtubule coupling. The Wordeman lab will study how the activities of microtubule regulatory enzymes implicated in kinetochore function are modulated through interactions with plus end binding proteins (+TIPs) and other microtubule binding factors. The Wordeman lab will also observe nucleotide turnover in a single microtubule-depolymerizing enzyme (MCAK) to test whether it enzymatically removes multiple tubulin subunits before detaching from the filament. These projects will bring us closer to a complete understanding of spindle function by elucidating the mechanisms underlying attachment of chromosomes to spindle microtubules, correction of attachment errors, and regulation of chromosome and spindle movements. Ultimately, having a mechanistic understanding of the spindle promises to revolutionize the design of chemotherapeutic drugs that target spindle components.